Modulation of pleiotrophin signaling by receptor-type protein tyrosine phosphatase beta/ç

ABSTRACT

The mechanism by which pleiotrophin binds to the receptor protein tyrosine phosphatase β/ζ (RPTP β/ζ) is disclosed along with methods of modulating both pleiotrophin expression and signaling to treat, prevent and inhibit abnormal cell growth states. Specifically provided are methods of inhibiting tumor growth, promotion, metastasis, invasiveness and angiogenesis as well as methods of preventing or inhibiting cell adhesion.

[0001] This application claims priority to copending U.S. provisionalpatent application Ser. No. 60/185,653, filed Feb. 29, 2000,incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Pleiotrophin (PTN) is a platelet-derived growth factor-inducibleheparin-binding growth and differentiation factor that signals diversephenotypes in normal and deregulated cellular growth anddifferentiation. See Milner, et al., (1989) Biochem. Biophys. Res.Commun. 165, 1096-1103; Rauvala, H. (1989) EMBO J. 8(10), 2933-2941; Liet al. (1990) Science 250, 1690-1694; Li et al., (1992) Biochem.Biophys. Res. Commun. 184: 427-432. PTN is nearly 50% identical with theretinoic acid-inducible factor midkine, which is also a growth anddifferentiation factor active in cultured fibroblasts, endothelial cellsand epithelial cells. See Li et al., 1990, supra; Muramatsu etal.,(1993) Dev. Biol. 159, 392-402. Pleiotrophin gene expression islimited to specific cell types at different times during development;however, in adults, pleiotrophin gene expression is constitutive andlimited to only a few cell populations except in sites of injury, whenits expression is sharply increased. See Li et al. 1990, supra; Li etal., 1992 supra; Silos-Santiago et al, (1996) J. Neurobiol. 31, 283-296;Yeh et al., (1998) J. Neurosci. 18: 3699-3707.

[0003] PTN also signals transformation; stable expression of anexogenous Ptn gene transforms NIH 3T3 cells and the Ptn-transformed NIH3T3 cells form rapidly growing highly vascularized tumors in nude mice.Chauhan et al., (1993) PNAS USA 90: 679-682. Significantly, high levelexpression of the Ptn gene is found in many different human malignanttumors and in the cell lines that have been derived from these tumors;however, Ptn gene expression is not found in the normal cells from whichthe malignancy is derived. Fang, Hartmann et al. 1992, J. Biol. Chem.267: 25889-97; Wellstein, Fang et al. 1992 J. Biol. Chem. 267: 2582-87;Tsutsui, Kadomatsu et al. 1993 Cancer Res. 53: 1281-85; Czubayko, Riegelet al. 1994, J. Biol. Chem. 269: 21358-63; Czubayko, Schulte et al.1995, Breast Cancer Res Treat 36: 157-68; Czubayko, Schulte et al. 1996PNAS USA 93: 14753-58; Brodeur, Nakagawara et al. 1997 J. Neurooncol.31: 49-55; Zhang, Zhong et al. 1997 J. Biol. Chem. 272: 16733-36; Zhangand Deuel 1999 Curr Opin Hematol 6:4450. Furthermore, high levelexpression of the Ptn gene may play a important role in developing amore aggressive phenotype in cancerous cells. Since it has also beenshown that interruption of endogenous PTN signaling by a dominantnegative PTN effector or a specific ribozyme reverses the malignantphenotype of human breast cancer cells (Zhang et al. 1997, J. Biol.Chem. 16733-36) and human melanoma cells (Czukayko et al., 1994 J. Biol.Chem. 269: 21358-63; Czubayko et al., 1996 PNAS USA 93: 14753-58),acquisition of PTN signaling during the course of these malignancies maytrigger a more aggressive phenotype.

[0004] It is known that cells rely, to a great extent, on extracellularmolecules as a means by which to receive stimuli from their immediateenvironment. These extracellular signals are important in the regulationof diverse cellular processes such as differentiation, contractility,secretion, cell division, cell migration, contact inhibition andmetabolism. The extracellular molecules include, for example, hormones,growth factors or neurotrrnsmitters, which may function as ligands thatbind specific cell surface receptors. The binding of these ligands totheir receptors triggers signal transduction, a cascade of reactionsthat brings about both the amplification of the original stimulus andthe coordinate regulation of the separate cellular processes mentionedabove.

[0005] A central feature of signal transduction is the reversiblephosphorylation of certain proteins. The phosphorylation ordephosphorylation of certain amino acid residues may triggerconformational changes in regulated proteins which results in thealteration of their biological properties. Proteins are phosphorylatedby protein kinases and are dephosphorylated by protein phosphatases.Phosphorylation is a dynamic process involving competing phosphorylationand dephosphorylation reactions, and the level of phosphorylation at anygiven instant reflects the relative activities, at that particularinstant, of the protein linases and phosphatases that catalyze thesereactions.

[0006] Protein linases and phosphatases are classified according to theamino acid residues they act on, for example, the class of tyrosinekinases and phosphatases act on tyrosine residues. See Fischer, E. H. etal., (1991) Science 253: 401-406; Schlessinger, J. and Ullrich, A.,(1992) Neuron 9:383-391; Ullrich, A. and Schlessinger, J., (1990) Cell61:203-212. Protein kinases and phosphatases may further be defined asbeing receptors, i.e., the enzymes are an integral part of atransmembrane, ligand-binding molecule, or as non-receptors, meaningthey respond to an extracellular molecule indirectly by being acted uponby a ligand-bound receptor.

[0007] The receptor class of protein tyrosine phasphatases (PTPs) ismade up of high molecular weight, receptor-linked PTPases, termedRPTPases. Structurally resembling growth factor receptors, RPTPasesconsist of an extracellular, putative ligand-binding domain, a singletransmembrane segment, and an intracellular catalytic domain (reviewedin Fischer et al., (1991) Science 253:401-406). Since the initialpurification, sequencing and cloning of a protein tyrosine phosphatase(Thomas, M. L. et al., (1985) Cell 41:83), additional potential proteintyrosine phosphatases have been identified. One such example is aproteoglycan-type protein tyrosine phosphatase, named protein tyrosinephosphatase ζ/receptor-like PTP β (RPTP β/ζ). Recently, PTN was found tointeract with the transmembrane RPTP β/ζ. See Maeda et al., (1996) J.Biol. Chem. 271: 21446-21452; Maeda, N. & Noda, M. (1998) J. Cell Biol.142, 203-216; Milev et al., (1998) J. Biol. Chem. 273: 6998-7005.

[0008] The PTN gene is a protooncogene and is expressed in many humantumors such as breast cancer, neuroblastoma, glioblastoma, prostatecancer, lung cancer and Wilms' tumor and cell lines derived from humantumors. See Fang et al., (1992) J. Biol. Chem. 267: 25889-25897; Chauhanet al, (1993) Proc. Natl. Acad. Sci. USA 90: 679-682; Wellstein et al.,(1992) J. Biol. Chem. 267: 2582-2587; Tsutsui et al., (1993) Cancer Res.53:1281-1285; Nakagawara et al., (1995) Cancer Res. 55: 1792-1797. Theimportance of PTN in malignant cell growth was first established whenintroduction of the exogenous Ptn gene into NIH 3T3 cells and NRK cellsled to morphological transformation, anchorage independent growth andtumor formation with significant neovascularization in vivo in the nudemouse. See Chauhan et al., 1993 PNAS USA 90: 679-82. It was subsequentlyshown that SW13 cells transformed by pleiotrophin also develop highlyvascular tumors in the flanks of athymic nude mice. See Fang et al.,1992 J. Biol. Chem. 267: 258889-97. Further, interruption of PTNsignaling has resulted in the reversal of the transformed phenotype ofhuman breast cancer cells that constitutively express the PTN gene(Zhang et al., (1997) J. Biol. Chem. 272: 16733-16736) and effectivelyreverted the malignant phenotype of cultured human melanoma cells(Czubayko et al, (1994) J. Biol. Chem. 269: 21358-21363). It is believedthat expression of the Ptn gene and its signaling pathway play a crucialregulatory role in many neoplasms of diverse origins. Thus,identification of the molecules and mechanisms of the PTN signalingpathway that are specific and crucial for tumor proliferation,angiogenesis and invasiveness would allow for the development ofclinical applications and specific anti-tumor drugs to treat cancer. Assuch, a need presently exists for the identification of compounds oragents that disrupt or interfere with PTN signaling in order toinfluence malignant transformation and inhibit tumor growth andangiogenesis.

[0009] Further, PTN also induces neurite outgrowth from neurons (Rauvala1989, supra; Li et al. 1990, supra) and glial process outgrowth fromglial progenitor cells, suggesting that Ptn gene expression mayinfluence a very broad range of functional activities. Since thepleiotrophin gene expression is upregulated by PDGF, PTN may actdownstream of PDGF to mediate aspects of the PDGF signal. Thus, theactivation of their respective signaling pathways is critical to thetemporal maturation of oligodendrocyte progenitors and the properties ofPTN suggest that PTN is ideally positioned to signal activation of genesimportant in maturation of glial elements at this critical time ofdevelopment. As differentiation of oligodendrocytes is required formyelination of nerve fibers and consequently, important to nerveconduction, the determination of mechanisms for modulating PTN signalingduring the differentiation of oligodendrocytes would be desirable.Accordingly, a need presently exists to determine the mechanism andmolecules by which PTN signals in order to develop methods to treat andprevent nerve injury and demyelinating diseases.

[0010] While the molecules through which PTN signals have not to datebeen established, in addition to interacting with RPTP β/ζ, PTN has alsobeen shown to bind to heparin, heparin sulfate proteoglycans andextracellular matrix. See Milner et al. 1989, supra; Rauvala, 1989supra; Li et al., 1990, supra; Raulo et al., (1994) J. Biol. Chem. 269:12999-13004; Maeda et al., (1996) J. Biol. Chem. 271: 21446-21452;Kinnunen et al., (1996) J. Biol. Chem. 271: 2243-2248. In addition tointeracting with RPTP β/ζ, PTN induces tyrosine phosphorylation of a 190kDa protein in PTN treated murine fibroblasts. See Li, Y. S. & Deuel, T.F. (1993) Biochem. Biophys. Res. Commun. 195: 1089-1095.

[0011] Thus, the interruption of PTN signaling impacts the eventsdownstream in the signaling cascade such as cell proliferation anddifferentiation. Accordingly, there is presently a need to understandPTN signaling and the interaction between RPTP β/ζ and PTN in order tomodulate the PTN signaling pathway to produce increased or decreased PTNactivity in order to define compounds which useful in therapy andtreating disease influenced by the expression of pleiotrophin such ascancer.

SUMMARY OF THE INVENTION

[0012] Applicants have shown that receptor protein tyrosine phosphataseβ/ζ (RPTP β/ζ) is the receptor for pleiotrophin (PTN). Binding of RPTPβ/ζ and PTN inhibits RPTP β/ζ enzymatic activity and results in higherlevels of tyrosine phosphorylation of β-catenin. Further, binding ofRPTP β/ζ and PTN also reduces the levels of the β-catenin interactionwith E-cadherin and thus affects the potential for cells to adhere witheach other.

[0013] The elucidation of this relationship between RPTP β/ζ and PTN canbe used to define compounds which useful in therapy and treatingdisease. For example, this pathway can be modulated to mimic increasedPTN activity in order to promote glial process formation, neuron growthand differentiation, endothelial cell growth and differentiation, andfibroblast growth. The method of accomplishing these effects involvesthe use of agents which either (a) mimic PTN binding to RPTP β/ζ, (b)inhibit the binding of RPTP β/ζ to B-catenin, (c) enhance or increasethe binding or the amounts of phosphorylated β-catenin to LEF-1 to forma transcription factor, or (d) mimic PTN binding to RPTP β/ζ.

[0014] Thus, among the several aspect of the present invention,therefore, include methods of monitoring levels of tyrosine phosphataseactivity of RPTP β/ζ in a cell or tissue comprising contacting the cellor tissue with an effective amount of pleiotrophin which binds to theactive site of RPTP β/ζ thereby reducing tyrosine phosphatase activityof RPTP β/ζ. Preferably, the administration of pleiotrophin results inthe increase of PTN activity and the reduction of tyrosine phosphataseactivity of RPTP β/ζ. Furthermore, the binding of pleiotrophin to theactive site of RPTP β/ζ preferably results in ligand-dependentdimerization of RPTP β/ζ and inactivates the catalytic activity of RPTPβ/ζ.

[0015] Another aspect of the present invention is directed to methods ofregulating levels of tyrosine phosphatase activity of protein tyrosinephosphataseζ/receptor-like protein tyrosine phosphatase β (RPTP β/ζ) ina cell or tissue, the method comprising:

[0016] a. determining whether the tyrosine phosphatase activity shouldbe reduced or increased in the cell or tissue to effectuate a desiredphysiologic change;

[0017] b. administering an effective amount of pleiotrophin,pleiotrophin inhibitor or mimic to reduce or increase the tyrosinephosphatase activity of RPTP β/ζ;

[0018] c. monitoring the cell or tissue for the appearance of thedesired physiologic change; and

[0019] d. determining whether to further modify levels of tyrosinephosphatase activity.

[0020] Yet another aspect of the present invention is directed tomethods of increasing tyrosine phosphorylation of β-catenin in a cell ortissue comprising contacting the cell or tissue that expresses RPTP β/ζwith an effective amount of pleiotrophin thereby reducing tyrosinephosphatase activity of RPTP β/ζ and increasing tyrosine phosphorylationof β-catenin.

[0021] In another aspect, methods for modulating cell-cell adhesion areprovided which include contacting a cell with pleiotrophin in an amountsufficient to inactivate tyrosine phosphatase activity of RPTP β/ζthereby increasing tyrosine phosphorylation of β-catenin in the cell anddecreasing interaction of β-catenin and E-cadherin.

[0022] The PTN signaling pathway can also be modulated to mimic reducedPTN activity to prevent or inhibit the growth or promotion of tumorcells and the loss of cell-cell interactions in cancer. This could beaccomplished by agents that (a) reduce or block PTN binding to RPTP β/ζ,(b) ensure the binding of RPTP β/ζ to β-catenin and its ability tomaintain normal steady state levels of tyrosine phophorylation ofβ-catenin, (c) reduce or eliminate the binding of phosphorylatedβ-catenin to LEF-1 to form a transcription factor, or (d) reduce oreliminate the translocation of phosphorylated β-catenin to the nucleus.Agents with these activities can be identified by screening chemicallibraries in in vitro assays as described in the Examples herein.

[0023] Thus, another aspect of the present invention is directed tomethods of inhibiting tumor invasiveness in a tissue comprisingcontacting the tissue with an effective amount of a compound which bindsto RPTP β/ζ or pleiotrophin thereby preventing pleiotrophin from bindingto RPTP β/ζ and decreasing tyrosine phosphatase activity of RPTP β/ζ.

[0024] Relatedly, the invention is further directed to methods ofinhibiting metastasis of a tumor comprising contacting the tumor with aneffective amount of a compound which binds to pleiotrophin or RPTP β/ζin the tissue thereby preventing pleiotrophin from binding to RPTP β/ζand increasing tyrosine phosphatase activity of RPTP β/ζ.

[0025] Yet another method of inhibiting tumor angiogenesis, progressionor promotion includes reducing the level of PTN signaling through RPTPβ/ζ in the tumor cells.

[0026] Further, methods of inhibiting tumor growth in a mammal includeadministering to the mammal an effective amount of a compound whichbinds to pleiotrophin or RPTP β/ζ thereby reducing the level ofpleiotrophin signaling through RPTP β/ζ in the tumor cells.

[0027] Also provided are pleiotrophin mimics which are compounds whichbind to pleiotrophin or RPTP β/ζ in manner which corresponds to theeffective binding of PTN to RPTP β/ζ in a cell or tissue.

[0028] Other aspects and features will be in part apparent and in partpointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIGS. 1A, 1B and 1C are a set of three western blots showing theassociation of RPTP β/ζ with PTN. FIG. 1A shows lysates of U373-MGglioblastoma cells immunoprecipitated with anti-RPTP β/ζ monoclonalantibodies. The immunoprecipitates were separated on 6% acrylamide gel,transferred to a poly(vinylidene difluoride) membrane, and probed withanti-RPTP β/ζ antibodies. The arrowheads indicate the RPTP β/ζ-splicedproducts of ≈230, 130 and 85 kDa. FIG. 1B shows Western analysis of RPTPβ/ζ captured by PTN-Fc. Lysates of U373-MG cells were incubated withPTN-Fc and proteins interactive with PTN-Fc (right lane) were capturedwith Protein A Sepharose-4B beads for 2 hours. The beads were washed incold lysis buffer, boiled in SDS/PAGE sample buffer, and the elutedproteins were separated on an 8% acrylamide gel and analyzed by Westernblots probed with anti-RPTP β/ζ monoclonal antibodies. As a control,PTN-Fc was replaced with an equal amount of human IgG (left lane). Thearrowheads indicate the ≈130 and ≈85 kDa-spliced products of RPTP β/ζ.FIG. 1C shows western analysis of RPTP β/ζ captured by endogenous PTN.Lysates of U373-MG cells were incubated with anti-PTN monoclonalantibodies (right lane) and the complexes were captured with Protein ASepharose-4B beads for 2 hours. The beads were washed in cold lysisbuffers, boiled in SDS-PAGE sample buffer, and the eluted proteins wereseparated on an 8% acrylamide gel and analyzed by Wester blots probeswith anti-RPTP β/ζ monoclonal antibodies. As a control, mouse IgGreplaced the anti-PTN antibody (left lane). The arrowheads indicate the130 and =85 kda-spliced products of RPTP β/ζ.

[0030]FIGS. 2A, 2B and 2C are a set of three bar charts showingPTN-dependent inhibition of the intrinsic tyrosine phosphatase activityof RPTP β/ζ. FIG. 2A shows inhibition of the endogenous RPTP β/ζtyrosine phosphatase activity in PTN-treated U373-MG cells. The left barrepresents tyrosine phosphatase activity in immunoprecipitates fromlysates of untreated cells with mouse IgG (control) to replace theanti-RPTP β/ζ antibodies. The center bar represents tyrosine phosphataseactivity in immunoprecipitates with anti-RPTP β/ζ antibodies fromlysates of untreated cells, and the right bar represents tyrosinephosphatase activity of immunoprecipitates with anti-RPTP β/ζ antibodiesfrom lysates of cells treated with recombinant PTN (50 ng/ml). FIG. 2Bshows inhibition of recombinant RPTP β/ζ phasphatase activity in Sf9cell membranes. The right two bars show membrane fractions of Sf9 cellsthat were infected by a baculovirus containing a cDNA-encoding RPTP β/ζ,or were uninfected (left two bars) that were untreated (−PTN) or treated(+PTN) with 50 ng/ml PTN. FIG. 2C shows a time course of PTN-dependentinactivation of RPTP β/ζ in PTN-treated (50 ng/ml) Sf9 cell membranesexpressing RPTP β/ζ (solid bars) and SF9 cell membranes without RPTP β/ζ(open bar, t=0 only).

[0031]FIGS. 3A and 3B are a set of two (FIG. 3A) and one (FIG. 3B)Western blots, respectively, showing physical and functional associationof β-catenin with PTN/RPTP β/ζ. FIG. 3A shows that PTN-Fc is in complexwith RPTP β/ζ and β-catenin. PTN-Fc treated confluent U373-MG cells from60-mm dish were chemically cross-linked with 3,3′dithiobissulfosuccinmidyl propionate. Lysates from PTN-Fc-treated, chemicallycross-linked cells (lanes 1) or Fc-(alone) treated (control) U373-MGcells (lane 2) were incubated with Protein A Sepharose, washed, elutedwith SDS sample buffer with 5% 2-mercaptoethanol, and analyzed in 6% SDSgels and Western blots. Lysates from untreated U373-MG cells alone (lane3) were also analyzed as a control. Western blots were analyzed withanti-β-catenin (right) or anti-RPTP β/ζ antibodies (left). Arrowheadsidentify RPTP β/ζ-spliced products of =250, 230, 180 and 85 kDa (left)and β-catenin (94 kDa) (right). FIG. 2B shows that β-catenin interactswith proximal (catalytic) domain of RPTP β/ζ. The GST-D1-RPTP β/ζwild-type, GST-D1-Cys-1925-Ser (inactivating) mutant fusion protein orGST alone were expressed and immobilized with glutathione-Sepharose-48beads, incubated with U373-MG cell lysates, washed, and analyzed inWestern analysis with the a-phosphotyrosine antibodies and visualizedwith the enhanced chemiluminescence ECLPLUS system (lower). The sameblot was reprobed with α-β-catenin antibodies and detected as above(upper).

[0032]FIGS. 4A and 4B are a pair of western blots showing increasedβ-catenin tyrosine phosphorylation. FIG. 4A is a time course of thetyrosine phosphorylation of β-catenin in response to PTN-FC treatment.Cells were treated with 10 ng/ml PTN-Fc for the times indicated. Lysateswere immunoprecipitated with α-β-catenin antibodies and analyzed inWestern blots probed with α-phosphotyrosine anti9bodies (upper) and theblots were reprobed with α-β-catenin antibodies and (lower). In FIG. 4B,U373-MG cells were treated with different doses of PTN-Fc for 20minutes. Cells were grown to near confluence, and then wereserum-starved for 48 hours. PTN-FC was added up to the indicatedconcentrations. The Fc fragment alone (20 ng/ml) was added as a control.Lysates were immunoprecipitated and analyzed in Western blots withantiphosphotyrosine antibodies as described above. Parallel immunoblotswere probed for β-catenin.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The following detailed description is provided to aid thoseskilled in the art in practicing the present invention. Even so, thisdetailed description should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

[0034] All publications, patents, patent applications, databases andother references cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference were specifically andindividually indicated to be incorporated by reference.

[0035] Abbreviations and Definitions

[0036] As used herein, “Ptn” refers to the pleiotrophin gene.

[0037] As used herein, PTN refers to the pleiotrohin protein.

[0038] The phrase “preventing or inhibiting interaction between RPTP β/ζand PTN” indicates that the normal interaction between a RPTP β/ζ andPTN is being affected either by being inhibited or reduced to such anextent that the binding of PTN to RPTP β/ζ is measurably lower than isthe case when PTN is interacting with RPTP β/ζ at conditions which aresubstantially identical (with regard to pH, concentration of ions, andother molecules) to the native conditions in the cell or tissue.

[0039] By the phrase “effective amount” is meant the amount of a desiredcompound necessary to (a) inhibit PTN binding to RPTP β/ζ and inhibitsits intrinsic catalytic activity, (b) inhibit the binding of RPTP β/ζ toβ-catenin, (c) inhibit the binding of phosphorylated β-catenin to LEF-1to form a transcription factor, or (d) mimic PTN binding to RPTP β/ζ.

[0040] By the term “a mimic of PTN” denotes any substance which mimicsor has the ability to bind to pleiotrophin or to RPTP β/ζ in a mannerwhich prevents the effective binding of PTN to RPTP β/ζ in a cell ortissue. Such a mimic of PTN can be a modified form of the intact PTN orit can be a modified form of the protein which may be coupled to aprobe, marker or another moiety. Another such mimic can be obtained bymodifying or mutating PTN so that it differs from the wild-type sequenceencoding PTN by the substitution of at least one amino acid residue ofthe wild-type sequence with a different amino acid residue and/or by theaddition and/or deletion of one or more amino acid residues to or fromthe wild-type sequence. The additions and/or deletions can be from aninternal region of the wild-type sequence and/or at either or both ofthe N- or C-termini. In the present context, PTN and mimics thereofexhibit at least one binding characteristic relevant for the interactionof PTN and RPTP β/ζ during PTN signaling in a cell or tissue. Suchmimics and compounds can also be small molecules which have the effectsof the mimicking factor described above.

[0041] The term “ligand-dependent receptor inactivation of RPTP β/ζ”refers to the mechanism in PTN signaling pathways by which PTN binds toRPTP β/ζ, inactivates the catalytic tyrosine phosphatase activity ofRPTP β/ζ and disrupts the normal role of RPTP β/ζ in the regulation ofsteady-state tyrosine phosphorylation of downstream signaling molecules.

[0042] In accordance with the present invention, applicant hasidentified that PTN is a natural ligand for RPTP β/ζ. PTN is the firstnatural ligand identified for any of the RPTP family and itsidentification provides a unique tool to pursue both the novel signalingpathway activated by PTN and the relationship of PTN signaling withother pathways regulating β-catenin. Furthermore, the finding of RPTPβ/ζ as the functional receptor for PTN is particularly interestingbecause to date, there are no known soluble ligands for this class oftransmembrane receptor tyrosine phosphatases and thus, PTN may be aunique probe for exploring the receptor class of transmembrane tyrosinephosphatases and how they signal.

[0043] Without intending to be bound by any particular theory, it isbelieved that PTN signals through “ligand-dependent receptorinactivation” of RPTP β/ζ and disrupts its normal roles in theregulation of steady-state tyrosine phosphorylation of downstreamsignaling molecules. Specifically, PTN binds to RPTP β/ζ, inducingligand-dependent dimerization of RPTP β/ζ and functionally inactivatesthe catalytic tyrosine phosphatase activity of RPTP β/ζ, presumablydenying the access of substrate(s) to its catalytic site. An activesite-containing domain of RPTP β/ζ both binds β-catenin and functionallyreduces its levels of tyrosine phosphorylation when added to lysates ofpervanidate-treated cells. Thus, this mechanism of PTN signaling throughRPTP β/ζ provides further insight into the mechanism by which PTN affectdownstream signaling.

[0044] Further, β-catenin interacts with the catalytically active D1domain of RPTP β/ζ and addition of the D1 domain of RPTP β/ζ with anactive tyrosine phosphatase catalytic site to lysates of cellspreviously treated with pervanidate sharply reduces levels of tyrosinephosphorylation of β-catenin; it is believed that β-catenin is asubstrate for the tyrosine phosphatase activity of RPTP β/ζ.Furthermore, because PTN rapidly signals tyrosine phosphorylation ofβ-catenin in intact U373-MG cells, inactivation of RPTP β/ζ is believedto be directly responsible for the increase in tyrosine phosphorylationof β-catenin as a result of the disruption of the normal balance oftyrosine kinase and phosphatase activities. Thus, RPTP β/ζ isintrinsically active and a principal regulator of tyrosinephosphorylation levels of β-catenin. In PTN-stimulated cells, RPTP β/ζis believed to be functionally inactivated, steady-state levels ofβ-catenin tyrosine phosphorylation and other downstream signalingmolecules are increased and a PTN-dependent downstream signaling cascadeis initiated. Thus, β-catenin not only is an endogenous substrate forRPTP β/ζ, but also a downstream mediator of PTN signaling.

[0045] Accordingly, the elucidation of this relationship between RPTPβ/ζ and PTN can be used to define compounds which useful in therapy andtreating disease. For example, this pathway can be modulated to mimicincreased PTN activity in order to promote glial process formation,neuron growth and differentiation, endothelial cell growth anddifferentiation, and fibroblast growth. The method of accomplishingthese effects involves the use of agents which either (a) mimic PTNbinding to RPTP β/ζ, (b) inhibit the binding of RPTP β/ζ to β-catenin,(c) enhance or increase the binding or the amounts of phosphorylatedβ-catenin to LEF-1 to form a transcription factor, or (d) mimic PTNbinding to RPTP β/ζ.

[0046] Thus, one aspect of the present invention provides methods ofregulating and/or modifying levels of tyrosine phosphatase activity ofRPTP β/ζ in a cell or a tissue. Such methods include determining whetherthe tyrosine phosphatase activity should be reduced or increased in thecell or tissue to effectuate a desired physiologic change; administeringan effective amount of pleiotrophin, pleiotrophin inhibitor orpleiotrophin mimic to reduce or increase the tyrosine phosphataseactivity of RPTP β/ζ; monitoring the cell or tissue for the appearanceof the desired physiologic change; and determining whether to furthermodify levels of tyrosine phosphatase activity. Such desiredphysiological changes include but are not limited to tumor promotion,growth angiogenesis, metastasis, modulation of cell-cell adhesion anddifferentiation of oligodendrocytes.

[0047] In another embodiment, the present invention provides methods formonitoring tyrosine phosphatase activity of RPTP β/ζ in a cell ortissue. This method involves contacting the cell or tissue with aneffective amount of pleiotrophin which binds to RPTP β/ζ, preferably theactive site of RPTP β/ζ. Preferably, in cells which express PTN,adminstering pleiotrophin results in the reduction of tyrosinephosphatase activity of RPTP β/ζ. Furthermore, the binding ofpleiotrophin to the active site of RPTP β/ζ preferably results inligand-dependent dimerization of RPTP β/ζ and inactivates the catalyticactivity of RPTP β/ζ.

[0048] Further, an (inactivating) active-site mutant of RPTP β/ζ alsobinds β-catenin but fails to reduce tyrosine phosphorylation ofβ-catenin. In parallel to its ability to inactivate endogenous RPTP β/ζ,PTN increases tyrosine phosphorylation of β-catenin in PTN-treatedcells. Thus, in unstimulated cells, RPTP β/ζ is intrinsically active andfunctions as an important regulator in the reciprocal control of thesteady state tyrosine phosphorylation levels of β-catenin by tyrosinekinases and phosphatases. As such, it is believed that RPTP β/ζ is afunctional receptor for PTN and that PTN signals throughligand-dependent receptor inactivation of RPTP β/ζ to increase levels oftyrosine phosphorylation of β-catenin to initiate downstream signaling.

[0049] Formation of cell-cell adhesion requires members of thecadherin-catenin families to link the highly conserved cadherincytoplasmic domain to the actin-based cytoskeleton and to connectadjacent cells via the cadherin extracellular domains. See Kypta et al.,(1996) J. Cell Biol. 134: 1519-1529; Tonks, N. K. & Neel, B. G. (1996)Cell 87: 365-368; Miller, J. R., & Moon, R. T. (1996) Genes Dev. 10,2527-2537. Balsamo et al. (J. Cell. Biol. 134: 801-813 (1996))demonstrated that the association of β-catenin with E-cadherin isinversely related to tyrosine phosphorylation levels of β-catenin inpervanidate-treated cells, raising the distinct possibility that throughits ability to increase tyrosine phosphorylation of β-catenin, PTNdisrupts the normal association of β-catenin and E-cadherin,underscoring the need for reciprocal control of tyrosine phosphorylationof β-catenin. Kypta et al.,(1996) J. Cell Biol. 134: 1519-1529;Hoschuetzky et al.,(1994) J. Cell Biol. 127:1375-1380; Fischer etal.,(1991) Science 253: 401-406; Brady-Kalnay et al.,(1995) J. Cell.Biol. 130: 977-986; Brady-Kalnay et al., (1998) J. Cell. Biol. 141:287-296. Because constitutive expression of PTN itself transforms cellswith striking loss of contact inhibition, cell adhesion, and strikingdisruption of cytoskeletal architecture, it is believed that the abilityof PTN to disrupt the reciprocal control of tyrosine phosphorylation ofβ-catenin by tyrosine kinases and phosphatases may account for many ofthe properties of PTN-transformed cells and those human cancer cellswhich constitutively express PTN.

[0050] Thus, in a preferred embodiment of the invention, tyrosinephosphorlyation of β-cateninin a cell or tissue is increased, preferablyin tissues or cells that express PTN. Preferably, increasing levels oftyrosine phosphorlyation of β-catenin in a cell or tissue reduces thelevel of β-catenin interaction with E-cadherin thus affecting cell-celladhesion. Loss of cell-cell interactions in cancer have a profoundeffect on tumor formation, promotion, angiogenesis and metastatsis.Preferably, increasing the levels of tyrosine phosphorlyation ofβ-catenin in a cell or tissue reduces the level of β-catenin interactionwith E-cadherin and more preferably, affects the potential for cells toadhere with each other. Hence, in a preferred embodiment, methods ofincreasing the levels of tyrosine phosphorlyation of β-catenin in a cellor tissue by administering pleiotrophin will affect cell-cell adhesion,preferably, by increasing the levels of tyrosine phosphorlyation ofβ-catenin to prevent or inhibit cell-cell adhesion. These methodsinvolve contacting the cell or tissue with an effective amount ofpleiotrophin thereby reducing tyrosine phosphatase activity of RPTP β/ζand increasing tyrosine phosphorylation of β-catenin. Preferably,pleiotrophin inactivates tyrosine phosphatase activity of RPTP β/ζ bybinding to the active site of RPTP β/ζ and more preferably, inducesligand-dependent dimerization of RPTP β/ζ. In a preferred embodiment,the ligand-dependent dimerization of RPTP β/ζ inhibits the ability ofβ-catenin to bind to the catalytic site of RPTP β/ζ, preferably the D1site of RPTP β/ζ.

[0051] In another aspect of the invention, the PTN signaling pathway canbe modulated to mimic reduced PTN activity thereby impacting eventsdownstream in the signaling cascade such as inhibiting the growth,proliferation, promotion, angiogenesis and/or metastatsis of tumor cellsand the loss of cell-cell interactions in cancer. This can beaccomplished by agents that (a) reduce or block PTN binding to RPTP β/ζ,(b) ensure the binding of RPTP β/ζ to β-catenin and its ability tomaintain normal steady state levels of tyrosine phophorylation ofβ-catenin, (c) reduce or eliminate the binding of phosphorylatedβ-catenin to LEF-1 to form a transcription factor, or (d) reduce oreliminate the translocation of phosphorylated β-catenin to the nucleus.Further, it is believed that PTN also signals nuclear translocation andtransactivation of genes signaling oncogenic pathways as a consequenceof the release of β-catenin from E-cadherin. Desirable results ofdecreasing PTN signaling is the reduction or inhibition of tumor growth,promotion, proliferation, angiogenesis and metastasis. During the studyof PTN signaling, Applicant observed that PTN not only transformed NIH3T3 cells but that NIH-PTN cells established rapidly growing highlyvascularized tumors in nude mice, suggesting that PTN may promote tumorgrowth by inducing tumor angiogenesis. Subsequently, it was shown thatintroduction of PTN into human adrenal carcinoma cells increased thenumber of new blood vessels when these cells were implanted into theflanks of nude mice. Furthermore, it was also possible to show that thestimulation of angiogenesis in SW13 cells by constitutive expression ofPtn could be localized to a domain of PTN within PTN amino acid residues69-136.

[0052] Accordingly in a preferred embodiment of the invention, methodsof inhibiting tumor invasiveness in a tissue the method includecontacting the tissue with an effective amount of a compound which bindsto RPTP β/ζ or pleiotrophin thereby preventing pleiotrophin from bindingto RPTP β/ζ and decreasing tyrosine phosphatase activity of RPTP β/ζ.

[0053] In another embodiment, methods are provided which reduce thelevel of PTN signaling through RPTP β/ζ in the tumor cells thusresulting in the inhibition of tumor angiogenesis, progression orpromotion.

[0054] Yet another embodiment provides a method of inhibiting tumorgrowth in a mammal comprising administering to the mammal an effectiveamount of a compound which binds to pleiotrophin or RPTP β/ζ therebyreducing the level of pleiotrophin signaling through RPTP β/ζ in thetumor cells. Preferably, the tumor cells are tumor cells from breastcancer, neuroblastoma, glioblastoma, prostate cancer, lung cancer andWilms' tumor.

[0055] In the above methods, an effective amount of a compound whichbinds to RPTP β/ζ or pleiotrophin can be antibodies to RPTP β/ζ,antibodies to pleiotrophin or a pleiotrophin mimic. Accordingly, in oneaspect, the present invention directed to a compound, preferably apleiotrophin mimic, which will mimic the capability of pleiotrophin tobind to RPTP β/ζ, thereby modulating, disrupting or interfering with PTNsignaling. The compound can be any compound, preferably a peptide, whichwill bind to pleiotrophin or RPTP β/ζ and prevent the binding ofpleiotrophin to RPTP β/ζ. In a preferred embodiment, the pleiotrophinmimic is a peptide compound. It will be appreciated that by virtue ofthe present invention, the polypeptide pleiotrophin mimic can besynthesized using conventional synthesis procedures commonly used by oneskilled in the art. For example, the polypeptides can be chemicallysynthesized using an automated peptide synthesizer (such as onemanufactured by Pharmacia LKB Biotechnology Co., LKB Biolynk 4170 orMilligen, Model 9050 (Milligen, Millford, Mass.)) following the methodof Sheppard, et al., Journal of Chemical Society Perkin I, p. 538(1981). In this procedure, N,N′-dicyclohexylcarbodiimide is added toamino acids whose amine functional groups are protected by9-flourenylmethoxycarbonyl (Fmoc) groups and anhydrides of the desiredamino acids are produced. These Fmoc-amino acid anhydrides can then beused for peptide synthesis. A Fmoc-amino acid anhydride corresponding tothe C-terminal amino acid residue is fixed to Ultrosyn A resin throughthe carboxyl group using dimethylaminopyridine as a catalyst. Next, theresin is washed with dimethylformamide containing piperidine, and theprotecting group of the amino functional group of the C-terminal acid isremoved. The next amino acid corresponding to the desired peptide iscoupled to the C-terminal amino acid. The deprotecting process is thenrepeated. Successive desired amino acids are fixed in the same manneruntil the peptide chain of the desired sequence is formed. Theprotective groups other than the acetoamidomethyl are then removed andthe peptide is released with solvent.

[0056] Alternatively, the polypeptides can be synthesized by usingnucleic acid molecules which encode the peptides of this invention in anappropriate expression vector which include the encoding nucleotidesequences. Such DNA molecules may be readily prepared using an automatedDNA sequencer and the well-known codon-amino acid relationship of thegenetic code. Such a DNA molecule also may be obtained as genomic DNA oras cDNA using oligonucleotide probes and conventional hybridizationmethodologies. Such DNA molecules may be incorporated into expressionvectors, including plasmids, which are adapted for the expression of theDNA and production of the polypeptide in a suitable host such asbacterium, e.g., Escherichia coli, yeast cell or mammalian cell.

[0057] It is known that certain modifications can be made withoutcompletely abolishing the polypeptide's ability to bind to pleiotrophinor RPTP β/ζ. Modifications include the removal and addition of aminoacids. Polypeptides containing other modifications can be synthesized byone skilled in the art and compounds comprising such polypeptides may betested for biological activity in the various assays and methodsdescribed in a later section. Thus, the effectiveness of thepolypeptides can be modulated through various changes in the amino acidsequence or structure.

[0058] Further, it should be understood that the mimic may be modifiedusing methods known in the art to improve binding, specificity,solubility, safety, or efficacy. A necessary characteristic of thesepreferred compounds is the capability to interact with pleiotrophin orRPTP β/ζ in such a manner that PTN signaling is disrupted or interferingprevented or inhibited.

[0059] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention, therefore all matter set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

EXAMPLES Example 1 Materials and Methods

[0060] Cell Culture

[0061] U373-MG glioblastoma (American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, USA) cells were used inall experiments and cultured in DMEM and 10% FCS unless otherwise noted.

[0062] Western Blot Analysis

[0063] U373-MG glioblastoma cells (≈10⁶) were lysed in 50 mM Tris-HCl(pH 8.0)/150 mM NaCl/1 mM EDTA/1% Triton X-100/1 mM phenylmethylsulfonylfluoride/0.5 μg/ml leupeptin/1 μM pepstatin/1 μg/ml aprotinin for 30minutes at 4° C., boiled in SDS/PAGE sample buffer (25 mM Tris-HCl, pH6.8/2.5% SDS/2.5% glycerol/5% 2-mercaptoethanol), separated by SDS/PAGE,transferred to poly(vinylidenedifluoride) membranes, probed withantibodies as indicated, and illuminated with the enhancedchemiluminescence ECL-PLUS system (Amersham Corp., Arlington Heights,Ill., USA).

[0064] Chemical Cross-Linking

[0065] U373-MG cells (≈10⁶) were incubated with the PTN-Fc fragment ofIgG (PTN-Fc) for 30 minutes at 37° C., washed with PBS, and incubatedwith 1 mM of the reversible cross-linking agent 3,3′-dithiobissulfosuccinimidyl propionate (Pierce Chemical Co., Rockford, Ill., USA)for 30 minutes at 37° C., and lysed.

[0066] PTN-Fc “Capture”

[0067] U373-MG cells (≈10⁶) were lysed as above, and proteins associatedwith PTN-Fc were bound to Protein A Sepharose-4B and, after washing,eluted by boiling in sample buffer and analyzed in Western blots asabove.

[0068] Glutathione S-Transferase (GST) “Capture” Assays

[0069] The GST-juxtamembrane (D1) fragment and the D1 fragmentCys-1925-Ser were prepared by using a human RPTP β/ζ cDNA fragment toencode amino acids 1655-2018 fused with GST in the expression plasmidPGEX-KG XhoI and XbaI sites. The constructs (or GST alone) wereexpressed in BL-21 competent cells from 5 ml overnight cultures, and therecombinant proteins were immobilized with 100 μl ofglutathione-Sepharose-4B beads (Amersham Pharmacia). The beads were thenincubated with U373-MG cell lysates from 60-mm confluent dishes, washed,eluted, and analyzed in Western blots as above.

[0070] Antibodies and Other Reagents

[0071] α-β-catenin and α-RPTP β/ζ antibodies were obtained fromTransduction Laboratories (Lexington, Ky.), and α-phosphotyrosinemonoclonal antibodies (4G10) were obtained from Upstate Biotechnology(Lake Placid, N.Y., USA). Recombinant PTN was purchased from SIGMAChemical Company (St. Louis, Mo., USA), and recombinant PTN-Fc waspurified from conditioned media of human embryonic kidney 293 cellsexpressing a cDNA that encodes the full-length PTN molecule fused at itsC terminus with the Fc fragment of IgG. For the dose responses of bothPTN and PTN-Fc to inactivate RPTP β/ζ, see below. PTN was used at 50ng/ml and PTN and PTN-Fc were established by using tyrosinephosphorylation of β-catenin and the ability PTN-Fc was used at 5 ng/ml,saturating levels of each, respectively.

Example 2 RPTP β/ζ Tyrosine Phosphatase Activity

[0072] PTN-treated U373-MG cells. Confluent U373-MG cells were incubatedeither with DMEM alone or 50 ng/ml recombinant PTN (Sigma) at 37° C. for15 minutes, washed three times with PBS, lysed as described above, andcleared at 14,000×g for 15 minutes 4° C. Equal amounts of lysates wereincubated with α-RPTP β/ζ antibodies or mouse IgG (control) at 4° C.overnight, incubated with Protein A Sepharose-4B at 4° C. for 2 hours,and washed three times in lysis buffer and once in assay buffer (20 mMimidazole, pH 7.2/0.1 mg/ml BSA). The phosphatase activity of theimmobilized RPTP β/ζ protein was assayed as follows: 50 μl of eitherRPTP β/ζ or mouse IgG immobilized on protein-A beads was added to theassay buffer, the reaction mixture (25 mM imidazole, pH 7.2/0.1 mg/mlBSA/10 mM DTT/100 nM ³²P-labeled substitute Raytide) was added to afinal volume of 80 μl, incubated at 30° C. for various times,terminated, and the ³²P released was quantitated by a charcoal-bindingassay. The synthetic peptide Raytide (Oncogene Science, Inc., Uniondale,N.Y., USA) was phosphorylated at its unique tyrosine residue byfollowing the manufacturer's instructions.

[0073] RPTP β/ζ activity in Sf9 cell membranes. The Bac-to-BacBaculovirus Expression System (Life Technologies, Gibco/BRL,Gaithersburg, Md., USA) was used to express RPTP β/ζ in Sf9 cells. Afull-length human RPTP β/ζ cDNA was cloned into a pFastBac donor plasmidat NotI and XbaI sites (pFastBac-RPTP β/ζ), transformed into DH10BacEscherichia coli which contains bacmid and helper virus, and plasmid DNAprepared. Sf9 cells were infected by the recombinant virus according tothe manufacturer's instructions. To prepare membrane fractions, cellswere sonicated in a hypotonic lysis buffer (25 mM Tris-HCl, pH 7.5/25 mMsucrose/0.1 mM EDTA/5 mM MgCl₂/5 mM DTT/1 mM phenylmethylsulfonylfluoride/0.5 μg/ml leupeptin/1 μg/ml aprotinin), nuclei were removed bylow-speed centrifugation, and membrane fractions were obtained bycentrifugation at 100,000×g for 60 minutes at 4° C. The resultingpellets were suspended by sonication in lysis buffer, brought to aconcentration of 2 mg/ml, and used to measure PTPase activity as above.

[0074] The assays were linear with time and protein concentration.

Example 3 Both Exogenous PTN and the Endogenous Ptn Gene ProductInteract with RPTP β/ζ

[0075] PTN-Fc was incubated with lysates of serum starved, confluentU373-MG cells, and proteins associated with PTN-FC were captured onProtein A Sepharose and probed by Western blot with anti-(α)-RPTP β/ζantibodies (FIG. 1B). Three major and other minor alternative-splicedforms of the single RPTP β/ζ gene have been identified (28, 29, 30), andthe results of the PTN-FC capture were therefore compared with Westernblots of immunoprecipitates from untreated U373-MG cell lysatesincubated with A-RPTP β/ζ antibodies (FIG. 1A). Major bands of ≈230,≈130, ≈85, and variably, in other experiments, ≈250 kDa were identified(FIG. 1A), consistent with the known different spliced forms of RPTP β/ζpreviously identified. Depending on the conditions of cell growth,different (presumably alternative-spliced) forms were identified. InWestern blots of proteins captured by PTN-Fc from U373-MG cell lysates,two major bands of ≈130 and 85 kDA were identified (FIG. 1B), suggestingthat PTN-Fc preferentially associates with isoforms of ≈130 and ≈85 kDa.However, when the blots were exposed for longer times, a faint band at230 kDa was also seen. When IgG alone was substituted for PTN-Fc, RPTPβ/ζ was not captured by Protein A Sepharose (FIG. 1B, left lane). Whenblots were reprobed with α-IgG antibodies that recognize the Fc portionof PTN-Fc or anti-PTN antibodies, it was established that PTN-Fc waspresent in the complex captured by Protein A Sepharose.

[0076] PTN itself is also expressed in U373-MG cells. To show that theendogenously expressed PTN and RPTP β/ζ interact with each other invivo, untreated U373-MG cell lysates were immunoprecipitated with α-PTNantibodies and analyzed in Western blots. Anti-RPTP antibodiesrecognized protein bands at 130 and 85 kDa (faint) (FIG. 1C). Theseresults thus establish that both exogenous and endogenous PTN physicallyinteract with the major alternatively spliced products of RPTP β/ζ inU373-MG cells.

Example 4 PTN Inactivates RPTP β/ζ Activity in vivo and in vitro

[0077] To directly determine if PTN affects the function of endogenousRPTP β/ζ, lysates of PTN-treated and control, untreated U373-MGglioblastoma cells were immunoprecipitated with α-RPTP β/ζ antibodies,incubated with Protein A Sepharose, and directly assayed for proteintyrosine phosphatase activity as described above (FIG. 2A). The effectsof PTN on the catalytic activity of recombinant RPTP β/ζ also weretested by using membrane fractions prepared from Sf9 insect cellsinfected with a baculovirus expressing recombinant RPTP β/ζ. Remarkably,the protein tyrosine phosphatase activity of the endogenous RPTP β/ζ inimmunoprecipitates from PTN-treated cells was reduced by more than 90%when compared with RPTP β/ζ from untreated cells and when corrected fornon-specific background (IgG controls, FIG. 2A). PTN also strikinglyreduced the catalytic activity of recombinant RPTP β/ζ in Sf9 membraneswhen background phosphatase activity was again corrected (FIG. 2B). Theinhibition by PTN is specific, because PTN inhibits tyrosine phosphataseactivity only in S19 cell membranes that express RPTP β/ζ and theinhibition is rapid (FIG. 2C). Nearly 70% of the phosphatase activity orRPTP β/ζ is lost in 5 minutes. Thus, PTN not only physically associateswith RPTP β/ζ but functionally, PTN profoundly reduces the catalyticactivity of RPTP β/ζ. Furthermore, because PTN effectively reduces theendogenous RPTP β/ζ activity, it can be concluded that RPTP β/ζ is anintrinsically activity tyrosine phosphatase, thereby suggesting thatRPTP β/ζ may be an important regulator of steady-state tyrosinephosphorylation of compatible intracellular substrates that themselvesare regulated by an intrinsically active tyrosine kinase activity.

Example 5 β-Catenin is a Potential Substrate for RPTP β/ζ

[0078] β-catenin is known to associate with other RPTPs and bephosphorylated in tyrosine (31, 32). β-catenin also is an importantsignaling molecule in development and in the wnt/APC−/− oncogenicpathways (33, 34, 35), suggesting that its signaling properties may beinfluenced by tyrosine phosphorylation and potentially be regulated byRPTP β/ζ and/or PTN. PTN-Fc-treated U373-MG cells were thereforeincubated with 3,3′-dithiobis-sulfosuccinimidyl-propionate and lysed.Proteins cross-linked to PTN-FC were captured with Protein A Sepharoseand analyzed by Western blot with either α-β-catenin (FIG. 3A, right) orα-RPTP β/ζ antibodies (control, FIG. 3A, left). In SDS/PAGE gels, ahigher molecular weight complex with very limited migration wasidentified, and immunoreactive PTN and RPTP β/ζ were identified in thisband. In other control experiments, both β-catenin (FIG. 2A Right, lane3) and RPTP β/ζ (FIG. 3A Left, lane 3) were readily recognized inuntreated U373-MG cell lysates. When the captured protein complex fromPTN-Fc treated cells cross-linked with3,3′-dithiobis-sulfosuccinimidyl-propionate was reduced before SDS/PAGEand analyzed in Western blots probed with α-RPTP β/ζ antibodies, RPTPβ/ζ-spliced forms of ≈130 kDa, and more weakly, ≈230 kDa, wereidentified (FIG. 3A, Left, lane 1). These forms were not identified inlysates of cells treated with the Fc fragment of IgG alone (FIG. 3Aleft, lane 2). Remarkably, PTN-Fc also captured β-catenin, based onrecognition by α-β-catenin antibodies and the migration of the bandrecognized by α-β-catenin antibodies and the migration of the bandrecognized by α-β-catenin at the estimated molecular mass of β-catenin(≈94 kDa) (FIG. 3A Right lane 1). β-catenin was not captured when cellswere treated with the Fc fragment of IgG alone (FIG. 3A Right, lane 2).The results confirm that the extracellular domain of RPTP β/ζ interactswith PTN-Fc and suggest that β-catenin interacts with its intracellulardomain. These results also raise the possibility that RPTP β/ζ links PTNsignaling to β-catenin.

[0079] RPTP β/ζ has two phosphatase domains in its C-terminalcytoplasmic tail. The juxtamembrane-proximal D1 domain of RPTP β/ζcontains an active tyrosine phosphatase catalytic unit whereas thejuxtamembrane-distal D2 domain lacks the required cysteine residue andthus is inactive (36). To see whether β-catenin associates with theactive site of RPTP β/ζ, the D1 domain and the D1 domain Cys-1925-Ser(active site inactivating) mutation were coupled with GST, incubated for15 minutes with U373-MG cell lysates from cells pretreated withpervanidate, and analyzed in Western blots. Both the active and inactiveD1 domains of RPTP β/ζ β-catenin (FIG. 3B, upper) at essentially equallevels. However, when the Western blots were reprobed withα-phosphotyrosine antibodies, the levels of tyrosine phosphorylation ofβ-catenin were sharply reduced in lysates incubated with the active (wt)D1 domain compared with the D1 domain Cys-1925-Ser (FIG. 3B, lower),localizing the association of β-catenin to the active site-containing D1domain and strongly suggesting that β-catenin is a substrate of RPTPβ/ζ.

Example 6 PTN Stimulates Tyrosine Phosphorylation of β-Catenin

[0080] To pursue the possibility that β-catenin is a substrate of RPTPβ/ζ in intact cells and that PTN-dependent inactivation of RPTP β/ζinfluences tyrosine phosphorylation of β-catenin, tyrosinephosphorylation was examined temporally after the addition of PTN-Fc tointact U373-MG cells. Tyrosine phosphorylation of β-catenin increasedwithin 2 minutes of addition of PTN and reached peak levels within 8minutes (FIG. 4A). The levels of β-catenin itself were essentiallyidentical, indicating that PTN-Fc had no detectable influence on thelevels of β-catenin protein. Furthermore, the response was PTN-Fcdose-dependent between 0.2 and 5 ng/ml (FIG. 4B).

[0081] All references, patents and patent applications are incorporatedherein by reference in their entirety. While this invention has beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method of regulating levels of tyrosinephosphatase activity of protein tyrosine phosphataseζ/receptor-likeprotein tyrosine phosphatase β (RPTP β/ζ) in a cell or tissue, themethod comprising: a. determining whether the tyrosine phosphataseactivity should be reduced or increased in the cell or tissue toeffectuate a desired physiologic change; b. administering an effectiveamount of pleiotrophin, pleiotrophin inhibitor or mimic to reduce orincrease the tyrosine phosphatase activity of RPTP β/ζ; c. monitoringthe cell or tissue for the appearance of the desired physiologic change;and d. determining whether to further modify levels of tyrosinephosphatase activity.
 2. A method of monitoring levels of tyrosinephosphatase activity of RPTP β/ζ in a cell or tissue, the methodcomprising contacting the cell or tissue with an effective amount ofpleiotrophin which binds to the active site of RPTP β/ζ thereby reducingtyrosine phosphatase activity of RPTβ/ζ.
 3. The method of claim 2wherein binding of pleiotrophin to the active site of RPTP β/ζ inducesligand-dependent dimerization of RPTP β/ζ thereby inactivating thetyrosine phosphatase activity of RPTP β/ζ.
 4. A method of increasingtyrosine phosphorylation of β-catenin in a cell or tissue, said methodcomprising contacting the cell or tissue that expresses RPTP β/ζ with aneffective amount of pleiotrophin thereby reducing tyrosine phosphataseactivity of RPTP β/ζ and increasing tyrosine phosphorylation ofβ-catenin.
 5. The method of claim 4 wherein pleiotrophin inactivatestyrosine phosphatase activity of RPTP β/ζ by binding to the active siteof RPTP β/ζ.
 6. The method of claim 5 wherein binding of pleiotrophin tothe active site of RPTP β/ζ induces ligand-dependent dimerization ofRPTP β/ζ.
 7. The method of claim 6 wherein ligand-dependent dimerizationof RPTP β/ζ inhibits the ability of β-catenin to bind to the catalyticsite of RPTP β/ζ.
 8. The method of claim 7 wherein catalytic site ofRPTP β/ζ is the D1 domain of RPTP β/ζ.
 9. A method of modulatingcell-cell adhesion, the method comprising contacting a cell withpleiotrophin in an amount sufficient to inactivate tyrosine phosphataseactivity of RPTP β/ζ thereby increasing tyrosine phosphorylation ofβ-catenin in the cell and decreasing interaction of β-catenin andE-cadherin.
 10. The method of claim 9 wherein pleiotrophin inactivatestyrosine phosphatase activity of RPTP β/ζ by binding to the active siteof RPTP β/ζ.
 11. The method of claim 10 wherein binding of pleiotrophinto the active site of RPTP β/ζ induces ligand-dependent dimerization ofRPTP β/ζ.
 12. The method of claim 11 wherein ligand-dependentdimerization of RPTP β/ζ inhibits the ability of β-catenin to bind tothe catalytic site of RPTP β/ζ thereby inhibiting tyrosinedephosphorylation of tyrosine residues of β-catenin.
 13. The method ofclaim 12 wherein catalytic site of RPTP β/ζ is the D1 domain of RPTPβ/ζ.
 14. A method of inhibiting tumor invasiveness in a tissue, themethod comprising contacting the tissue with an effective amount of acompound which binds to RPTP β/ζ or pleiotrophin thereby preventingpleiotrophin from binding to RPTP β/ζ and decreasing tyrosinephosphatase activity of RPTP β/ζ.
 15. The method of claim 14 wherein thecompound is an antibody to pleiotrophin.
 16. The method of claim 14wherein the compound is an antibody to RPTP β/ζ.
 17. The method of claim14 wherein the compound is a pleiotrophin mimic.
 18. A method ofinhibiting metastasis of a tumor, the method comprising contacting thetumor with an effective amount of a compound which binds to pleiotrophinor RPTP β/ζ in the tissue thereby preventing pleiotrophin from bindingto RPTP β/ζ and increasing tyrosine phosphatase activity of RPTP β/ζ.19. The method of claim 18 wherein the compound is an antibody topleiotrophin.
 20. The method of claim 18 wherein the compound is anantibody to RPTP β/ζ.
 21. The method of claim 18 wherein the compound isa pleiotrophin mimic.
 22. A method of inhibiting tumor angiogenesis,progression or promotion, said method comprising reducing the level ofPTN signaling through RPTP β/ζ in the tumor cells.
 23. The method ofclaim 22 wherein the level of PTN signaling is reduced by administeringan effective amount of antibodies to pleiotrophin to the cells of thetumor.
 24. The method of claim 22 wherein the level of PTN signaling isreduced by administering an effective amount of antibodies to RPTP β/ζto the cells of the tumor.
 25. The method of claim 22 wherein the levelof PTN signaling is reduced by administering a pleiotrophin mimic to thecells of the tumor.
 26. A method of inhibiting tumor growth in a mammal,the method comprising administering to the mammal an effective amount ofa compound which binds to pleiotrophin or RPTP β/ζ thereby reducing thelevel of pleiotrophin signaling through RPTP β/ζ in the tumor cells. 27.The method of claim 26 wherein the method comprises inhibiting cellularproliferation of the tumor cells.
 28. The method of claim 26 wherein themethod comprises inhibiting invasiveness of the tumor.
 29. The method ofclaim 26 wherein the method comprises inhibiting metastasis of thetumor.
 30. The method of claim 26 wherein the tumor cells are tumorcells from breast cancer, neuroblastoma, glioblastoma, prostate cancer,lung cancer and Wilms' tumor.
 31. The method of any one of claims 26-30wherein the compound is an antibody to pleiotrophin.
 32. The method ofany one of claims 26-30 wherein the compound is an antibody to RPTP β/ζ.33. The method of any one of claims 26-30 wherein the compound is apleiotrophin mimic.